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Shutian's Research

Week Fourteen

I did protein purification and characterization on Monday.

Figure 1: Absorbance of elution 1. The concentration is 45.

Figure 2: Absorbance of elution 2. The concentration is 10.80. This is much lower than the absorbance of elution 1 because most of the proteins was already washed into the elution 1 tube.

Figure 3: The protein band is at about 45 kDa. The protein PP2Ctg is supposed to be from 42 - 52 kDa. Even though the expression is relatively weak, the correct protein was expressed successfully.
Lane 1 – PageRuler Ladder
Lane 2 – Cell lysate before induction
Lane 3 – Cell lysate after induction
Lane 4 – soluble fraction
Lane 5 – Flow through (waste)
Lane 6 – Wash Lane
Lane 7 – Elution 1
Lane 8 – Elution 2

Figure 4: Top 10 ligands from two of the libraries. These fitness scores are pretty low. At the same time, none of these satisfied the Lipinski's Rule of Five.

Week Thirteen

I did my second run for the ChemBridge library.

Week Twelve

My virtual screening for ChemBridge- diversity3D.sdf is still running. I have been working on the virtual screening.

Week Eleven

I have been checking on my virtual screening, and the ChemBridge- diversity3D.sdf is still running. I have finished protein expression.
My pellet weight was 2.81.

Week Ten

This week, I did my PCR clean-up and saved the samples in -20 degree Celsius. I am also doing my protein expression. My virtual run for ChemBridge-diversity3D.sdf is still running.

Week Nine

I have been working on the Virtual Screening. I have done the 2nd run for the cb_306_3d.sdf and the HF9PlatesPlates5_9.sdf. However, the ChemBridge-diversity3D.sdf is still on its first run because it is such a large library.

Week Eight

I also ran the primary runs for the HF9 and the Chembridge libraries.

Primer Overlap Primer Design

tgpp 1	ATGGATGTACCTCCGACCATCCACGTACCGCTCCC	35
tgpp 2	GTCGGTGAAGATCGCCGCGTCGAACGCTGGGTAAGAGGTTGGCGGGAGCGGTACGTGGAT	60
tgpp 3	CGGCGATCTTCACCGACATCGGTGGCCGTAAACACCAGGAGGATCGTTTCACCCTGTGCC	60
tgpp 4	ACCGAAGAACGCGCAGTCGTCACGACCCGGAACCAGCTGTGGGCACAGGGTGAAACGATC	60
tgpp 5	ACTGCGCGTTCTTCGGTGTTTTTGATGGTACCGTTGGTGATTTCGCGTCTGAAAACGTTA	60
tgpp 6	ACGCCGGAGAAGAGATGAGCTGCGGTACAACCAGATCTTTAACGTTTTCAGACGCGAAAT	60
tgpp 7	TCATCTCTTCTCCGGCGTGGCAGGAAGTTACCGAAATGCTGCGTTCTGATGTTCCGGCAA	60
tgpp 8	CGCCTGGTCGAGGAGTTGCGGCAGTTTTTCGTCTACTTCAGTTGCCGGAACATCAGAACG	60
tgpp 9	AACTCCTCGACCAGGCGGTTGACGATATGTACAAAAACGCAGACAACGAACTGGTTAAAA	60
tgpp 10	GGTAGAAGACGCGTAGTCTTTGTTCAGCTGTTCGCACATTTTAACCAGTTCGTTGTCTGC	60
tgpp 11	AAAGACTACGCGTCTTCTACCTCTGTGACCGCTGTTCTGGCGAAAGGCTTCGTTGCGGTT	60
tgpp 12	GGTCTCAACACCCATGGCGATGCGAGAGTCACCGAGGTGACCAACCGCAACGAAGCCTTT	60
tgpp 13	GCCATGGGTGTTGAGACCCCGAATGGCCTGAACTGCGAGTTCCTCACCGTTGACCACAAA	60
tgpp 14	CCGTTACGCATGATGCGGAGCTTTTCGTGCGGCATGTCCGGTTTGTGGTCAACGGTGAGG	60
tgpp 15	CCGCATCATGCGTAACGGTGGTTCTGTTGAATACCTGCACAATCATAACAACAAACCTTT	60
tgpp 16	CGAGATTTACGGAAAGAGAAGTCACCACCACGGATGAAAGGTTTGTTGTTATGATTGTGC	60
tgpp 17	GACTTCTCTTTCCGTAAATCTCGTGGTGAACAGCCAATGCAACTGCAATACTCTCGTGCG	60
tgpp 18	TGGTTAGAGAGACCATACATTTTGAGGTCTTTACCACCGAACGCACGAGAGTATTGCAGT	60
tgpp 19	CAAAATGTATGGTCTCTCTAACCAGCCGGACGTTCGCGTTGTTCGTGTTACCCCGCAACA	60
tgpp 20	ATGACGTCCCAGAGACCGTCGGTCGCGAGGATCATTACACGATGTTGCGGGGTAACACGA	60
tgpp 21	CGGTCTCTGGGACGTCATGTCTGCTGCGCAAGCAGTCGAAATTGCTATGCAAGCGCGTCA	60
tgpp 22	CAGGGTCATTTCTACCAGCGCCTGGGCTGGGTTACGACCTTCCTGACGCGCTTGCATAGC	60
tgpp 23	CGCTGGTAGAAATGACCCTGGCGGAACAGCAGTCTCGCAATCAGTCCGCTGACAACACGG	60
tgpp 24	GTCGGTTTTCTTGAAAAAGACGGTCATGGCCGTGTTGTCAGCGGAC	46

Week Seven

I ran the primary and secondary PCR and they came out very well. I have also finished the Primer Design for the overlap.

Lane 1: Empty
Lane 2: 100 bp ladder
Lane 3: Primary PCR
Lane 4: Secondary PCR
Lane 5: Run over from the secondary PCR

Week Six

I have been working on the PCR primer overlap this week. I have stored the primary and secondary PCR in the -20 degrees refrigerator. The PCR squared still needs to be run.

Week Five

The Virtual Screening Refresher was done running. Through the analysis by using PyMOL, the top 10 ligands were observed and compared to MTX.

(1) Output 5, Ligand 1

(2) Output 1, Ligand 1

(3) Output 2, Ligand 1

(4) Output 1, Ligand 2

(5) Output 2, Ligand 2

(6) Output 2, Ligand 3

(7) Output 4, Ligand 1

(8) Output 3, Ligand 1

(9) Output 3, Ligand 2

(10) Output 5, Ligand 2

MTX

2D Images for the top 3 bind ligands

1)

Week Four

I was working on the Virtual Screening Refresher. The second run took a while because I was waiting for the blades.

Week Four

2nd PCR: I accidentally added DNA ladder into Sample A/lane 1. But the result was still able to show up for the sample.

I also finished the Pymol Refresher.

This is the 2H2Q molecule with the two chains specifically labeled.

3CLQ

RMS = 1.126 (111 to 111 atoms)

1U72

3HBB

Week Three

The results of the Submit DNA sequencing:

This is blasted against the human genome.

This is blasted against the protein.

RE Digest with the bands clearly shown.

Lane1: Empty
Lane 2: 1 kb ladder
Lanes 3 & 4: Uncut plasmid
Lane 5: PvuII
Lane 6: EcoRI
Lane 7: EcoRI and PvuII

PCR try #3

Lane 1: Empty
Lane 2: 100 bp Ladder
Lane 3: Sample A
Lane 4: Sample B
Lane 5: Sample C
Lane 6: Sample D

Week Two

Gel observed on September 9, 2011

RE Digest and PCR 2: The results are unclear because the gel was too soft. So, when the samples were put into the wells, the walls of the wells collapsed and the samples were mixed together.

Lane 1: EcoRI digest reaction (This was repeated due to the collapse of the wall)
Lane 2: 1 kb ladder
Lane 3: EcoRI digest reaction
Lane 4: PvuII digest reaction
Lane 5: EcoRI and PvuII digest reaction
Lane 6: Uncut plasmid (pGBR22 426.3 ng/ul)

Lane 7: 100 bp ladder
Lane 8: Sample A
Lane 9: Sample B
Lane 10: Sample C
Lane 11: Sample D
Lane 12: 1 kb ladder (This was repeated due the collapse of the wall)

This gel electrophoresis failed because the gel was not solidified enough. As a result, the walls of the wells collasped and the samples smeared together.